Increasing numbers of bacterial pathogens pose serious human health threats due to their acquisition of multidrug resistance. A common mechanism of resistance involves extrusion of drugs through multidrug efflux pumps. The Resistance-Nodulation-Cell Division (RND) multidrug efflux system is crucial to antibiotic resistance in numerous Gram-negative bacterial pathogens, including the opportunistic pathogens Pseudomonas aeruginosa and Haemophilus influenzae. RND efflux systems are also encoded by Escherichia coli O157:H7, Salmonella enterica, Shigella flexneri, and Yersinia pestis. The most thoroughly characterized RND system is the E. coli AcrAB-TolC system, which promotes efflux of novobiocin, erythromycin, fusidic acid, tetracycline, and chloramphenicol among others. Like all RND systems, AcrAB-TolC consists of an inner membrane protein, AcrB; a periplasmic protein, AcrA, which belongs to the so called family of membrane fusion proteins (MFP), although MFPs do not fuse membranes; and an outer membrane protein, TolC. Significantly, the structures of the inner and outer membrane proteins AcrB and TolC, respectively, are known, but there is no atomic resolution structure of the MFP AcrA to explain its essential and active role in drug efflux. A key next step in pursuing longer-term mechanistic studies on the role of the MFP AcrA and RND systems in general is to determine the structure of AcrA, and thereby fill in the structural picture of the AcrAB-TolC system. Our specific aims are to complete the X-ray crystal structure determination of AcrA, and to use this structural information to identify regions of AcrA that interact with AcrB and TolC through in vitro site-directed, cross-linking experiments. These short-term goals will enable the design of inhibitors of RND efflux pumps, which have the potential of rendering useful many antibiotics that have become clinically ineffective. [unreadable] [unreadable]